Virus-like particle (VLP) based small molecule-protein interaction trap

ABSTRACT

This disclosure relates to a virus-like particle in which a small molecule-protein complex is entrapped, ensuring the formation of the small molecule-protein complex under physiological conditions, while protecting the small molecule-protein complex during purification and identification. The disclosure further relates to the use of such virus-like particle for the isolation and identification of small molecule-protein complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S.application Ser. No. 16/532,154, filed Aug. 5, 2019, pending, which is acontinuation to U.S. application Ser. No. 15/559,748, filed Sep. 19,2017, issued, which is a national phase entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/EP2016/056331, filed Mar. 23, 2016,designating the United States of America and published in English asInternational Patent Publication WO 2016/150992 A1 on Sep. 29, 2016,which claims the benefit under Article 8 of the Patent CooperationTreaty to Great Britain Patent Application Serial No. 1504859.8, filedMar. 23, 2015, the disclosure of each of which is hereby incorporatedherein in its entirety by this reference.

TECHNICAL FIELD

The present application relates to a virus-like particle, in which asmall molecule-protein complex is entrapped, ensuring the formation ofthe small molecule-protein complex under physiological conditions, whileprotecting the small molecule-protein complex during purification andidentification. The application further relates to the use of suchvirus-like particle for the isolation and identification of smallmolecule-protein complexes.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS A TXT FILE

Pursuant to 37 C.F.R. § 1.821(c) or (e), files containing a TXT versionof the Sequence Listing have been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

BACKGROUND

Molecular interactions, such as protein-protein interactions, areessential components of virtually all cellular processes. The binding oftwo or more compounds in a cell can have a wide array of effects,including modulating signal transduction, regulating gene transcription,and promoting cellular replication or apoptosis. Several human diseasesare associated with malfunctioning of molecular interactions.

Researchers have developed several approaches in attempts to identifymolecular interactions. A major breakthrough in the detection ofprotein-protein interactions was obtained by the introduction of thegenetic approaches, of which the yeast two-hybrid (Fields and Song,1989) is the most important one. Although this technique became widelyused, it has several drawbacks. The fusion proteins need to betranslocated to the nucleus, which is not always evident. Proteins withintrinsic transcription activation properties may cause false positivesignals. Moreover, interactions that are dependent upon secondarymodifications of the protein such as phosphorylation cannot be easilydetected.

Several alternative systems have been developed to solve one or more ofthese problems.

Approaches based on phage display do avoid the nuclear translocation.WO9002809 describes how a binding protein can be displayed on thesurface of a phage, such as a filamentous phage, wherein the DNAsequence encoding the binding protein is packaged inside the phage.Phages, which bear the binding protein that recognizes the targetmolecule, are isolated and amplified. Several improvements of the phagedisplay approach have been proposed, as described, e.g., in WO9220791,WO9710330 and WO9732017.

However, all these methods suffer from the difficulties that areinherent to the phage display methodology: the proteins need to beexposed at the phage surface and are so exposed to an environment thatis not physiological relevant. Moreover, when screening a phage library,there will be a competition between the phages that results in aselection of the high affinity binders.

A major improvement in the detection of protein-protein interactions wasdisclosed in WO0190188, describing the so called Mappit system. Themethod, based on a cytokine receptor, allows not only a reliabledetection of protein-protein interactions in mammalian cells, but alsomodification-dependent protein interactions can be detected, as well ascomplex three hybrid protein-protein interactions mediated by a smallcompound (Caligiuri et al., 2006). However, although very useful, thesystem is limited in sensitivity and some weak interactions cannot bedetected. Moreover, as this is a membrane-based system, nuclearinteractions are normally not detected. Recently, a cytoplasmicinteraction trap has been described, solving several of thoseshortcomings (WO2012117031). However, all these “genetic” systems relyon the overexpression of both interaction partners, which may result infalse positive signals, due to the artificial increase in concentrationof the interaction partners.

As an alternative for the genetic protein-protein interaction detectionmethods described above, biochemical or co-purification strategiescombined with mass spectrometry (MS)-based proteomics (Paul et al.,2011; Gingras et al., 2007) can be used. For the co-purificationstrategies, a cell homogenate is typically prepared by a detergent-basedlysis protocol, followed by capture using a (dual) tag approach (Gavinet al., 2002) or via specific antibodies (Malovannaya et al., 2011). Theprotein complex extracted from the “soup” of cell constituents must thensurvive several washing steps, mostly to compensate for the sensitivityof contemporary MS instruments, before the actual analysis occurs. Thereare no clear guidelines on the extent of washing or on available buffersand their stringency. Most lysis and washing protocols are purelyempirical in nature and were optimized using model interactions. It is,therefore, hard to speculate on the loss of factors during these steps(false negatives), or the possibility of false interactions due to lossof cellular integrity (false positives). Use of metabolic labelingstrategies allows separation between the proteins sticking to thepurification matrix, and between the proteins that associatespecifically to the bait protein. Depending on the purificationconditions and the sensitivity of the MS instruments used, it is noexception to find more than 1000 proteins in the eluted fraction of agel-free AP-MS experiment (www.crapome.org).

The classical approach to identify target proteins for small moleculesrelies on the use of “purification handles” that are added to the smallmolecule. A biotin group is typically used to modify the small molecule,preferentially through a linker and on a permissive site of themolecule. The modified small molecule is then used to capture theassociated molecules by a classical pull-down approach usingstreptavidin beads on a lysate. In a recent implementation, Ong andcolleagues describe the use of quantitative proteomics based onmetabolic labeling (Stable Isotope Labeling of Amino acids in CellCulture—SILAC), to define the proteins that bind specifically to a smallmolecule. The authors use “small-molecule beads” that were prepared bydirect chemical coupling of the small molecules to the beads (Ong etal., 2009). Bantscheff and colleagues described a method wherein a panelof broad range kinase inhibitors was coupled to a matrix. This matrixwas then incubated with cell lysates to bind a significant portion ofthe kinome. By adding increasing concentration of candidate kinaseinhibitors, on- and off-target kinases can be identified (Bantscheff etal., 2007). A major limitation of this approach is the lack of broadspecificity inhibitors outside of the kinase family making it difficultto translate the strategy to other protein target families. In addition,off-targets outside of the kinase family are not readily identified.Another very recent development is thermal profiling to assess thechange in thermal stability of proteins upon binding of a smallmolecule. Proteins tend to aggregate depending on the temperature whichis affected by binding of ligands or posttranslational modifications.Savitski and colleagues performed this analysis in a proteome-widemanner using quantitative proteomic approaches and were able to identifyknown and novel targets for different small molecules (Savitski et al.,2014).

Recently, a co-purification technique has been disclosed in WO2013174999that allows for evaluating protein-protein interactions in theirphysiological environment. The complexes are trapped via the p55 GAGprotein into artificial virus-like particles (VLPs) that are budded fromhuman cells. The complexes are protected during the enrichment processin a so-called “Virotrap particle.” However, Virotrap, even in itsconditional mode of operation, does not identify previously unknownsmall molecule-protein interactions.

It would be advantageous to entrap small molecule-protein complexesunder physiological conditions and thereby evaluate physiologicallyrelevant small molecule-protein interactions.

BRIEF SUMMARY

To evaluate whether a solution could be found for isolating previouslyunidentified small molecule-protein interactions under physiologicallyrelevant conditions, different isolation protocols were evaluated.

Surprisingly, it was found that new methods derived from the recentlydescribed Virotrap protocol (WO2013174999) also can be used to trap asmall molecule together with its physiological binding partners intoVLPs that are budded from human cells. The very mild enrichment of thecomplex ensures the identification of relevant small molecule-proteininteractions in physiological environments.

According to a first aspect, provided herein are artificial virus-likeparticles (VLPs), comprising:

-   -   (a) a VLP-forming polypeptide;    -   (b) a fusion construct comprising two small molecules covalently        linked to each other, wherein the first small molecule interacts        with the VLP-forming polypeptide and the second small molecule        interacts with at least one polypeptide different from the        VLP-forming polypeptide; and    -   (c) a polypeptide interacting with the second small molecule of        (b).

According to particular embodiments, the VLP-forming polypeptide is afusion protein. According to further particular embodiments, theVLP-forming polypeptide is a fusion protein comprising the HIV p55 GAGprotein.

Also provided is the use of an artificial VLP for the detection of smallmolecule-protein interactions.

According to a further aspect, methods are provided for detecting smallmolecule-protein interactions, comprising:

-   -   (1) expressing a VLP-forming polypeptide in a cell;    -   (2) recruiting a fusion construct comprising two small molecules        covalently linked to each other, wherein the first small        molecule interacts with the VLP-forming polypeptide and the        second small molecule interacts with at least one polypeptide        different from the VLP-forming polypeptide, to the VLP-forming        polypeptide;    -   (3) allowing a polypeptide to interact with the second small        molecule of (2);    -   (4) isolating the VLPs; and    -   (5) analyzing the entrapped complex.

According to particular embodiments, the VLPs are isolated by anaffinity chromatography-based method.

According to specific embodiments, the entrapped complex is analyzedusing a mass spectrometry-based method.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: General overview of the small molecule-protein trapping method.Expression of a GAG-eDHFR fusion protein (1) (i.e., the VLP-formingpolypeptide) results in submembrane multimerization (2) and subsequentbudding of virus-like particles (VLPs) from cells (3). The VLPs can thenbe purified and analyzed by co-complex MS analysis (4) providing a wayof purifying protein complexes under physiological conditions. Theaddition of a small molecule fusion construct during VLP productionresults in binding of the small molecule fusion construct to theGAG-eDHFR fusion protein and to trapping of small molecule-interactingproteins in the VLPs.

FIG. 2: Scheme for the use of the VLP-forming polypeptide GAG-eDHFR totrap small molecule-protein interactions. As a non-limiting example, atarget interacting protein complex is recruited to the GAG-eDHFR fusionprotein via the small molecule fusion construct methotrexate(MTX)-PEG-simvastatin.

FIG. 3: Chemical structures of the different small molecule fusionconstructs used in this study.

FIG. 4: Dose-response curve of the MASPIT signal obtained for theinteraction between the MTX-PEG6-tamoxifen molecule and HSD17B4.Luciferase activity is expressed as fold induction compared to controlsamples treated without the small molecule fusion. Error bars representthe standard deviation of technical triplicates.

DETAILED DESCRIPTION Definitions

This disclosure will be described with respect to particular embodimentsand with reference to certain drawings but the disclosure is not limitedthereto, but only by the claims. Any reference signs in the claims shallnot be construed as limiting the scope. The drawings described are onlyschematic and are non-limiting. In the drawings, the size of some of theelements may be exaggerated and not drawn on scale for illustrativepurposes. Where the term “comprising” is used in the present descriptionand claims, it does not exclude other elements or steps. Where anindefinite or definite article is used when referring to a singularnoun, e.g., “a,” “an,” or “the,” this includes a plural of that noununless something else is specifically stated.

Furthermore, the terms first, second, third and the like in thedescription and in the claims, are used for distinguishing betweensimilar elements and not necessarily for describing a sequential orchronological order. It is to be understood that the terms so used areinterchangeable under appropriate circumstances and that the embodimentsof the disclosure described herein are capable of operation in othersequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in theunderstanding of the disclosure. Unless specifically defined herein, allterms used herein have the same meaning as they would to one skilled inthe art of this disclosure. Practitioners are particularly directed toSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Press, Plainsview, N.Y. (1989); and Ausubel et al.,Current Protocols in Molecular Biology (Supplement 47), John Wiley &Sons, New York (1999), for definitions and terms of the art. Thedefinitions provided herein should not be construed to have a scope lessthan understood by a person of ordinary skill in the art.

A “virus-like particle,” or “VLP,” as used here, is a particlecomprising at least a viral particle-forming polypeptide, but preferablywithout the viral DNA or RNA. “Virus-like particle-forming polypeptides”or “VLP-forming polypeptides,” as used here, are known to the personskilled in the art and are polypeptides or proteins that allow theassembly of viral particles, and preferably budding of the particles ofthe cell. A VLP-forming protein is sufficient to form a VLP, and therewill typically be more than one (typically identical) VLP-formingprotein in a VLP. According to specific embodiments, the VLP-formingpolypeptides are fusion proteins of a VLP-forming protein and anotherprotein, polypeptide or protein subunit.

It is particularly envisaged that the VLPs as described herein do notcontain viral genetic material, so they are non-infectious.

“Polypeptide” refers to a polymer of amino acids and does not refer to aspecific length of the molecule. This term also includes modificationsof the polypeptide, such as glycosylation, phosphorylation andacetylation of the naturally occurring amino acids, and includessubstitutions of one or more of the naturally occurring amino acids withnon-natural analogs.

The “fusion construct comprising two small molecules” as used hereincomprises two small molecules covalently linked to each other, whereinthe first small molecule functions as and is referred to as “recruitingelement” that interacts with the VLP-forming polypeptide. The secondsmall molecule functions as and is referred to as “bait” and interactswith at least one polypeptide different from the virus-likeparticle-forming polypeptide (the “prey” polypeptide).

“Small molecules” are low molecular weight organic compounds, having amolecular weight of 10,000 Daltons or less, of natural or syntheticnature.

“Interacts with” typically means, but is not limited to, “binds to.” Ofnote, interaction can be indirect as well.

The term “recruited to” in relation to the fusion construct that isrecruited to the VLP refers to allowing the recruiting element of thefusion construct to interact with and/or bind to the VLP-formingpolypeptide.

According to a first aspect, an artificial virus-like particle (VLP) isprovided, comprising:

-   -   (a) a VLP-forming polypeptide;    -   (b) a fusion construct comprising two small molecules covalently        linked to each other, wherein the first small molecule        (“recruiting element”) interacts with the VLP-forming        polypeptide and the second small molecule “bait”) interacts with        at least one polypeptide different from the VLP-forming        polypeptide; and    -   (c) a polypeptide (“prey”) interacting with the second small        molecule (“bait”) of (b).

The VLPs can be derived from numerous viruses. Examples of suchparticles have been described in the art and include, but are notlimited to particles derived from virus families including Parvoviridae(such as adeno-associated virus), Retroviridae (such as HIV),Flaviviridae (such as Hepatitis C virus), Orthomyxoviridae (such asInfluenza virus), and Rhabdoviridae (such as vesicular stomatitisvirus). The particles typically comprise viral structural proteins, suchas Envelope or Capsid, and result in the self-assembly of virus-likeparticles.

According to specific embodiments, the VLP-forming polypeptide is afusion protein. Thus, according to these embodiments, rather than takinga viral structural protein as such, the protein (or a functional partthereof) is fused to another polypeptide. As such, the viralparticle-forming polypeptide then comprises (or consists of) twodifferent polypeptide domains, typically (but not necessarily) takenfrom two different proteins.

According to particular embodiments, the VLP-forming polypeptide may bea modified form of the natural occurring VLP-forming protein, as long asthe modifications do not inhibit the particle formation. A modificationor functional fragment as used here is a modification or functionalfragment that is still capable of forming virus-like particles that arecapable of entrapping the small molecule-protein complex according tothe disclosure. Examples of modifications include, e.g., deletionsand/or mutations. Particularly envisaged are deletions and/or mutationsthat reduce the binding of the VLP-forming polypeptide with hostproteins with the objective to minimize the background protein list.

A particularly envisaged VLP-forming polypeptide is the HIV p55 GAGprotein. According to further particular embodiments, the VLP-formingpolypeptide is a fusion protein comprising the HIV p55 GAG protein.Preferably, the polypeptide fused to the p55 GAG protein comprisesdihydrofolate reductase (DHFR), even more preferably, E. colidihydrofolate reductase (eDHFR).

The artificial VLP contains a fusion construct comprising two smallmolecules covalently linked to each other. The two small moleculestypically are independently selected from compounds with a molecularweight of 10,000 Daltons or less. Particularly, the fusion constructcomprises two compounds with each a molecular weight of 5,000 Daltons orless. Particularly, the small molecule comprises compounds with amolecular weight of 2,000 Daltons or less. Particularly, the smallmolecule comprises compounds with a molecular weight of 1,000 Daltons orless and, most particularly, with a molecular weight of 500 Daltons orless.

As the recruiting element binds to the VLP-forming polypeptide, andthere typically is more than one VLP-forming polypeptide in a virus-likeparticle, there can be more than one fusion construct in the VLP.Typically, the fusion constructs comprising two small molecules areidentical in a VLP (so that the same prey proteins can be identified),but this need not be the case.

The fusion construct contains a small molecule acting as recruitingelement, and a small molecule acting as bait, wherein both molecules arecovalently connected. The nature and the length of the covalent linkerused in the fusion construct are not vital to the disclosure (as long asthey do not interfere with incorporation in the VLP). Particularlyenvisaged herein is the use of polyethyleneglycol (PEG) as a covalentlinker between the molecules in the fusion construct.

The small molecule recruiting element as used here is a compound thatrecruits, directly or indirectly, the small molecule bait together withits physiological binding partners into VLPs. Typically, this is done bybinding of the VLP-forming polypeptide. In other words, the smallmolecule recruiting element has an affinity for the VLP-formingpolypeptide, and is able to interact with (or bind to) the VLP-formingpolypeptide.

Since it is particularly envisaged that the VLP-forming polypeptide is afusion protein, the small molecule recruiting element does not need tohave an affinity for a native VLP-forming polypeptide, but can haveaffinity for a different protein. This allows greater flexibility in thechoice of recruiting element and VLP-forming polypeptide. According tothese embodiments, the fusion protein partner of the VLP-formingpolypeptide interacts with the recruiting element of the small moleculefusion.

Many small molecules that have affinity for a given protein are known tothe skilled person, and the nature of the binding pair (recruitingelement/fusion protein partner of the VLP-forming polypeptide) is notessential, on condition that the fusion protein still effectively formsVLPs. According to particular embodiments, the small molecule recruitingelement is selected from the group consisting of methotrexate (MTX) andtrimethoprim (TMP). According to a specific embodiment, the smallmolecule recruiting element is not MTX. According to a specificembodiment, the fusion construct comprising two small molecules is notan MTX-based fusion construct.

The other small molecule of the fusion construct, the bait molecule, isused to attract an interacting polypeptide. The binding partner of thebait molecule may be known (e.g., to confirm an interaction, forinstance for modified proteins) or unknown (e.g., in identifying targetsof a given small molecule drug). According to a specific embodiment, thebinding partner of the bait molecule (the prey protein) is unknown.According to a specific embodiment, the small molecule bait is known andthe interacting prey protein is unknown. The interaction between thesmall molecule and its prey protein can be covalent or non-covalent, andcan be direct or indirect. According to a specific embodiment, theinteraction between the small molecule bait and the interacting preyprotein is direct. According to a particular embodiment, the polypeptidebait is unknown and binds to a known small molecule bait.

According to a specific embodiment, an artificial VLP is provided,consisting of:

-   -   (a) a VLP-forming polypeptide;    -   (b) a fusion construct comprising two small molecules covalently        linked to each other, wherein the first small molecule        (“recruiting element”) interacts with the VLP-forming        polypeptide and the second small molecule (“bait”) interacts        with at least one polypeptide different from the VLP-forming        polypeptide; and    -   (c) a polypeptide (“prey”) interacting with the second small        molecule (“bait”) of (b).

According to a very specific embodiment, the small molecule bait is notFK506. Besides the small molecule fusion construct and the interactingpolypeptide, the virus-like particle may comprise other compounds,recruited to the small molecule-protein complex, wherein all thecompounds together form one complex. In a particular embodiment, the VLPas described above entraps two molecular interactions, i.e., themolecular interaction between the VLP-forming polypeptide and the fusionconstruct comprising two small molecules and the molecular interactionbetween the fusion construct and the prey polypeptide. In a veryspecific embodiment, the VLP as described above does not entrap threemolecular interactions, i.e., not more than the two molecularinteractions described above. In a very specific embodiment, themolecular interactions are non-covalent interactions. In a specificembodiment, the molecular interactions do not trigger posttranslationalmodifications, such as, but not limited to, phosphorylations. Accordingto some embodiments, the prey polypeptide interacting with the smallmolecule bait is a polypeptide isolated in physiological conditions,i.e., a naturally occurring polypeptide, or parts thereof, and not afusion protein. According to a particular embodiment, the preypolypeptide is a fusion protein, wherein an unknown prey polypeptide isfused to a known (fusion partner) protein and the fusion partner proteindoes not act as bait. According to some embodiments, the interactingpolypeptide is the sole interaction partner of the bait small moleculeof the fusion construct. However, it is also possible that more than onesingle protein is included in one VLP (e.g., if the fusion constructinteracts with more than one single protein), wherein each prey proteininteracts directly with the bait. According to some embodiments,complexes of two or more different proteins are included in one VLP,wherein the different proteins interact directly and/or indirectly withthe bait.

The prey protein can be an endogenous protein, can be provided bytransfection of expression plasmids or by RNA transfection in the cellproducing the VLPs, or can be added directly as a recombinant protein.

Of note, one bait small molecule may be able to bind to several preypolypeptides. Thus, when referring to a polypeptide (“prey”) interactingwith the second small molecule in the VLP, it is explicitly meant that“a” is one or more. Indeed, there can be different polypeptides in thesame VLP, even if only one type of fusion construct is used.

The VLP-forming polypeptide (or typically, a multitude thereof) forms aviral structure, consisting of a hollow particle, in which the smallmolecule fusion construct and the interacting (prey) polypeptides aretrapped. In particular embodiments, the small molecule fusion isanchored to the viral structure, ensuring the capturing of the complexformed by the small molecule fusion and the prey polypeptide into theinside of the virus-like particle. Preferably, the anchoring of thesmall molecule fusion is direct to the VLP-forming polypeptide, and doesnot comprise any independent linker molecule. An “independent linkermolecule,” according to the this disclosure, is a molecule that bindsnon-covalently to the viral particle-forming protein at one hand, and toa fusion protein at the other hand.

The VLPs provided herein are particularly useful for the detection ofsmall molecule-protein interactions, particularly in native and/orphysiological conditions. Accordingly, in another aspect of thedisclosure, the use of an artificial virus-like particle, as describedherein, is provided for the detection of small molecule-proteininteractions.

Still another aspect of the disclosure is a method for detecting smallmolecule-protein interactions, comprising (1) expressing a VLP-formingpolypeptide in a cell; (2) recruiting a fusion construct comprising twosmall molecules covalently linked to each other, wherein the first smallmolecule interacts with the VLP-forming polypeptide and the second smallmolecule interacts with at least one polypeptide different from theVLP-forming polypeptide, to the VLP-forming polypeptide; (3) allowing apolypeptide to interact with the second small molecule of (2) to thefusion construct; (4) isolating the VLPs; and (5) analyzing theentrapped complex. In a specific embodiment, the method as describedabove detects novel small molecule-protein interactions, i.e.,interactions of small molecules and proteins that have not beendescribed before. In a particular embodiment, the method as describedabove detects interactions between a known small molecule bait and anunknown protein. the unknown protein can be provided by, e.g., but notlimited to, a protein library that can be expressed from, but notlimited to, a cDNA library. In a specific embodiment, the proteinlibrary provides more than one potential binding partner for the smallmolecule bait. In a specific embodiment, the protein library providesmore than two potential binding partners for the small molecule bait. Ina specific embodiment, the protein library provides more than tenpotential binding partners for the small molecule bait. In a specificembodiment, the protein library provides more than 10² potential bindingpartners for the small molecule bait. In a specific embodiment, theprotein library provides more than 10³ potential binding partners forthe small molecule bait. In a specific embodiment, the protein libraryprovides more than 10⁴ potential binding partners for the small moleculebait. In a specific embodiment, the protein library provides more than10⁵ potential binding partners for the small molecule bait. In aspecific embodiment, the protein library provides more than 10⁶potential binding partners for the small molecule bait. In a particularembodiment, a single small molecule bait is used to screen proteinlibraries. In a particular embodiment, a single small molecule bait isused to screen cDNA libraries. In a particular embodiment, a singlesmall molecule bait is used to screen ORF libraries.

Preferably, the cell is a mammalian cell.

Steps 1-3 result in the generation of a VLP with a complex entrappedtherein. The order of steps 1-3 can differ. For instance, thepolypeptide interacting with the second small molecule may first bind tothe fusion construct prior to interaction of the first small molecule ofthe fusion construct with the VLP-forming polypeptide.

Isolating the VLPs can, e.g., be done by means of a purification tag. Atagged version (e.g., FLAG-tag) of the vesicular stomatitis virusglycoprotein (VSV-G), a transmembrane protein typically used forpseudotyping lentiviral vectors, can be expressed in the producer cellsto allow a simple one-step purification. Isolating is then typicallydone by affinity chromatography or a similar method using an antibodydirected against the tag (e.g., M2 anti-FLAG antibody). Antibodiesdirected against VSV-G itself or other molecules present on the surfacecan be used to enrich VLPs. Other methods of VLP enrichment includeultracentrifugation of supernatant containing VLPs, gradientcentrifugation of supernatant containing VLPs or precipitation of theVLPs from supernatant using precipitating agents. These methods requirea centrifugation step to pellet VLPs. These pellets can be re-dissolvedand processed for analysis. Filtration kits to enrich and purifyparticles are also available.

Typically, analysis of the entrapped complex will entail identificationof the polypeptide interacting with the second small molecule.Particularly, the analysis of the entrapped molecular complex is anMS-based analysis. If a particular polypeptide is expected, a westernblot analysis can be performed to confirm the presence of thepolypeptide in the particles. It is clear for the person skilled in theart that molecular interactions of any nature can be detected with themethod.

It is to be understood that although particular embodiments, specificconfigurations as well as materials and/or molecules, have beendiscussed herein for cells and methods according to this disclosure,various changes or modifications in form and detail may be made withoutdeparting from the scope and spirit of this disclosure. The followingexamples are provided to better illustrate particular embodiments, andthey should not be considered limiting the application. The applicationis limited only by the claims.

EXAMPLES

Materials and Methods

Generation of Plasmids

The p55 GAG fusion constructs were generated by PCR amplification of thep55 GAG coding sequence using primer 1 (5′-CTCTAAAAGCTGCGGGGCCCGCTAGCGCCACCATGGGTGCGAGAGCGTCAG-3′ (SEQ ID NO:1)) and primer 2(5′-TGTATTCGGTGAATTCTGAGCTCGTCGACCCGCCTTGTGACGAGGGGTCGCTGC-3′ (SEQ IDNO:2)) from the pCMV-dR8.74 packaging construct (Addgene) and bysubsequent In-Fusion™ reaction (Clontech) in pMG1-Ras, a Ras expressionvector used in the MAPPIT system (Eyckerman et al., 2001), resulting ina p55 GAG-RAS under control of the strong SRalpha promoter(pMET7-GAG-Ras). The pMD2.G pseudotyping vector, expressing VSV-G undercontrol of a CMV promoter, was kindly provided by Didier Trono (EPFL,Lausanne, Switzerland). The pcDNA3-FLAG-VSV-G and pcDNA3-Etag-VSV-G werecloned by introducing the respective epitope coding sequences in apermissive site in the extracellular part of VSV-G. The coding sequencefor the E. coli dihydrofolate reductase (eDHFR) was transferred into thepMET7-GAG plasmid from previous constructs (Risseeuw et al., 2013).Synthesis of the small molecule fusions was described before (Risseeuwet al., 2013; Lievens et al., 2014).

Production and Analysis of VLPs

For mass spectrometry, 10⁷ HEK293T cells were seeded in five flasks (75cm²) and transfected the next day with a total of 15 μg DNA per flaskusing polyethylene imine reagent. The following DNA quantities were usedper flask: 7.5 μg of GAG-eDHFR, 5.4 μg of mock vector and 2.1 μg of a50/50 mix of pMD2.G and pcDNA3-FLAG-VSV-G. The small molecule fusionswere added immediately after transfection to the producing cells at aconcentration of 1 μM. The same dilution of dimethyl sulfoxide (DMSO)was added in control experiments to control the effect of this chemicalon the cells. The cellular supernatant was harvested after 32 hours andwas centrifuged for 3 minutes at 450×g to remove cellular debris. Thecleared supernatant was then filtered using 0.45 μm filters (Millipore).A total of 100 μl MyOne™ Streptavidin T1 beads (Life Technologies)pre-loaded with 10 μg ANTI-FLAG® BioM2-Biotin antibody (Sigma-Aldrich®)was used to bind the tagged particles. Particles were allowed to bindfor 2 hours by end-over-end rotation. The total supernatant wasprocessed in three consecutive binding steps. Bead-particle complexeswere washed once with washing buffer (TWB: 20 mM Tris-HCl pH 7.5, 150 mMNaCl) and were then eluted with FLAG peptide (200 μg/ml in TWB) andlysed by addition of SDS to a final concentration of 0.1%. After 5minutes, SDS was removed using HiPPR Detergent Removal Spin Columns(Pierce, Thermo Scientific) followed by boiling and overnight digestionwith 0.5 μg sequence-grade trypsin (Promega). After acidification (0.1%TFA final), the peptides were separated by nano-LC and directly analyzedwith a Q Exactive instrument (Thermo Scientific). Searches wereperformed using the MASCOT algorithm (Version 2.4.1. Matrix Science) at99% confidence against human and bovine SWISSPROT accessions (Release2013_02) complemented with HIV-1, EGFP, VSV-G and FLAG-VSV-G proteinsequences.

Example 1: Viral Trapping of Simvastatin Binders

To screen for simvastatin binders, cells were transfected with theGAG-eDHFR construct and then treated immediately after transfection withthe MTX-PEG6-simvastatin molecule. A total of five biological repeatswere performed together with four DMSO control experiments. The list ofcandidate interactors was obtained after removal of all proteins(including proteins identified with a single peptide) that were found in19 control samples, the four DMSO samples and the other small moleculesamples (e.g., the protein list of the five simvastatin samples waschallenged against a total of 29 samples). The control samples are anumber of successful Virotrap experiments using unrelated proteins togenerate a list of proteins that can be considered background proteins,and that can be subtracted from the protein list obtained for the actualsamples.

For each small molecule condition, two lists of proteins (annotated bytheir gene name) were reported. The left part of the tables (“Allproteins”) contains all unique protein identifications (thus alsoproteins identified with a single peptide) obtained after removal of thebackground proteins. The right part of the tables (“Proteins identifiedwith >1 peptide”) contains only the proteins identified with highconfidence (at least two modified peptide identifications for eachprotein). Some recurrent proteins that were identified in multipleexperiments with a single peptide are removed from this list (e.g., PYGMidentified with low confidence in two repeat experiments).

For simvastatin, 3-hydroxy-3-methylglutaryl coenzyme A reductase (genename: HMGCR), the primary target for the statin family of molecules, wasidentified in all five biological repeat experiments. In addition,squalene epoxidase (SQLE) was identified with at least two peptides inthree experiments. This enzyme is an important downstream component ofthe sterol biosynthesis pathway, and may interact directly with HMGCR.UBIAD1, a known interaction partner of the HMGCR protein (Nickerson etal., 2013) was identified in one experiment with at least two peptides.SARM1 may be a novel target protein for simvastatin.

TABLE 1 Proteins identified with the MTX-PEG6-simvastatin small moleculefusion construct. All protein identifications (including single peptideidentifications) or identified proteins with at least two peptides areshown with their recurrent identification in five biological replicates.Recurrence Gene name (x/5) All proteins (incl. low confidenceidentifications) HMGCR 5 SQLE 4 SARM1 3 PYGM 2 AKT3 1 ATP6V0C 1 CNPY2 1CTU1 1 DBI 1 DDTL 1 EIF1AY 1 FABP1 1 FHL1 1 GALNT1 1 GINS1 1 KIAA0319L 1KRT6B 1 LAMTOR5 1 LNPEP 1 LOC512271 1 LOC524129 1 MOXD1 1 PDIA4 1 PRKCSH1 RAB4A 1 RBP4 1 SBDS 1 SH3GL2 1 SLC17A1 1 SLC46A1 1 TMED5 1 TMED9 1UBIAD1 1 VAMP4 1 VPS37D 1 Proteins identified with >1 peptide (highconfidence) HMGCR 5 SARM1 3 SQLE 3 CTU1 1 KIAA0319L 1 KRT6B 1 PDIA4 1UBIAD1 1

Example 2: Viral Trapping of Tamoxifen Binders

For tamoxifen, four biological repeats were performed where the cellswere treated with MTX-PEG6-tamoxifen after transfection. The obtainedprotein lists after viral trapping and MS analysis were challenged withprotein lists coming from the four DMSO controls, the 19 controlexperiments and the lists obtained for the MTX-PEG6-simvastatin andMTX-PEG6-reversine (see below) for a total of 30 samples.

The results were presented similarly as for the simvastatin example (seeExample 1) with a table containing gene name identifiers and recurrenceof detection in four experiments, for both high confidence and lowconfidence identifications (see Table 2).

TABLE 2 Proteins identified with the MTX-PEG6-tamoxifen small moleculefusion construct. All protein identifications (including single peptideidentifications) or identified proteins with at least two peptides areshown with their recurrent identification in four biological replicates.Recurrence Gene name (x/4) All proteins (incl. low confidenceidentifications) HSD17B4 4 REEP6 4 SSNA1 4 RNF5 3 TBL2 3 DCAKD 2 EI24 2REEP4 2 SEC61G 2 ATL2 1 C28H10ORF35 1 CALU 1 CGN1 1 CYP51A1 1 DNAJB12 1ECE1 1 ENPP4 1 EPHX1 1 ESYT1 1 ESYT2 1 FAM62A 1 GRAMD4 1 HSD17B12 1ILVBL 1 ITPRIPL1 1 LRRC59 1 MDH2 1 MINK1 1 NCS1 1 PCSK5 1 PRAF2 1 PRSS31 PTPLAD1 1 RTN3 1 RTN4 1 SEC61B 1 SEMA4G 1 SREBF2 1 SSR3 1 TAOK2 1TMPRSS13 1 UBQLN2 1 VAPA 1 WDR36 1 ZMPSTE24 1 Proteins identifiedwith >1 peptide (high confidence) HSD17B4 4 REEP6 3 REEP4 2 SSNA1 2 TBL22 ECE1 1 ESYT1 1 ESYT2 1 FAM62A 1 MDH2 1 RTN4 1 SEC61G 1 UBQLN2 1 VAPA 1

The interaction between tamoxifen and HSD17B4 was confirmed using abinary MASPIT assay (see Example 4).

Example 3: Viral Trapping of Reversine Binders

For reversine, two experiments were formed where cells were transfectedwith the GAG-eDHFR construct and then treated after transfection withMTX-PEG6-reversine during VLP production. After purification andproteomic analysis, the obtained lists were challenged with the combinedproteome list of the four DMSO and 19 unrelated control experiments, andof the MTX-PEG6-simvastatin and MTX-PEG6-tamoxifen lists.

TABLE 3 Proteins identified with the MTX-PEG6-reversine small moleculefusion construct. All protein identifications (including single peptideidentifications) or identified proteins with at least two peptides areshown with their recurrent identification in two biological replicates.Recurrence Gene name (x/2) All proteins (incl. low confidenceidentifications) IGF1R 2 INSR 2 NQO2 2 APMAP 1 BZW1 1 CAMKV 1 CTNNA2 1DYNC2H1 1 FKBP3 1 GAK 1 GEMIN5 1 HTRA3 1 LMAN1 1 MBTPS1 1 MFI2 1 MST1R 1MYO1D 1 NEK2 1 NT5C 1 PPM1L 1 RCL 1 RPS6KA1 1 TMEM30A 1 TMEM9 1 TMX1 1USP7 1 YIPF5 1 ZNF827 1 Proteins identified with >1 peptide (highconfidence) IGF1R 2 INSR 2 GEMIN5 1 NQO2 1 RCL 1

Example 4: Confirmation of the Interaction Between Tamoxifen and HSD17B4Using a MASPIT Assay

The binding of HSD17B4 to tamoxifen was further confirmed using theMASPIT technology. The binary MASPIT assay was essentially performed asdescribed before (Risseeuw et al., 2013). Briefly, HEK293T cells wereseeded in black tissue-culture treated 96-well plates at 10.000cells/well in 100 μl culture medium (DMEM supplemented with 10% fetalcalf serum), and grown at 37° C., 8% CO₂. Twenty-four hours later cellswere transfected with a combination of the pCLG-eDHFR plasmid (Risseeuwet al., 2013), the pMG1-HSD17B4 construct and thepXP2d2-rPAP1-luciferase reporter (Caligiuri et al., 2006). ThepMG1-HSD17B4 construct was generated by Gateway transfer of the fullsize HSD17B4 ORF, obtained as an entry clone in the hORFeome collection,into the Gateway compatible pMG1 prey destination vector as describedearlier (Lievens et al., 2009). Twenty-four hours after transfection,cells were either left unstimulated or treated with 100 ng/ml leptin,with or without addition of MTX-PEG6-tamoxifen. Another 24 hours later,luciferase activity was assayed using the Luciferase Assay System kit(Promega).

As shown in FIG. 4, the binary MASPIT assay showed increased luciferaseactivity in a dose-dependent manner upon addition of MTX-PEG6-tamoxifen,confirming the interaction of tamoxifen with HSD17B4.

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What is claimed is:
 1. A method for detecting a small molecule-proteininteraction, the method comprising: expressing a fusion proteincomprising a virus-like particle (VLP)-forming polypeptide; recruitingto the fusion protein a small molecule construct comprising a first anda second small molecule covalently linked to one another, wherein thefirst small molecule of the small molecule construct interacts with thefusion protein and the second small molecule of the small moleculeconstruct interacts with at least one polypeptide other than the fusionprotein; interacting the at least one polypeptide other than the fusionprotein with the second small molecule of the small molecule construct;forming and isolating VLPs comprising the fusion protein; and analyzingan entrapped complex in the isolated VLP.
 2. The method according toclaim 1, wherein the VLPs are isolated by affinity chromatography. 3.The method according to claim 1, wherein the entrapped complex isanalyzed by mass spectrometry.
 4. The method according to claim 1,wherein the VLP-forming polypeptide is a fusion protein comprising aVLP-forming polypeptide and a protein recruiting the first smallmolecule of the small molecule construct.
 5. The method according toclaim 4 wherein the fusion protein comprises HIV p55 GAG protein as theVLP-forming polypeptide.
 6. The method according to claim 1, wherein thepolypeptide interacting with the second small molecule of the smallmolecule construct is a physiological binding partner of the secondsmall molecule.
 7. The method according to claim 1, wherein the firstsmall molecule of the small molecule construct is not methotrexate. 8.The method according to claim 1, wherein the VLP-forming polypeptidefurther comprises a purification tag.
 9. The method according to claim6, wherein the fusion protein comprises HIV p55 GAG protein asVLP-forming polypeptide.